Pyk2 controls rab proteins by vesicle trafficking in glioblastoma cells

Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival of less than one year. GBM progression depends on constant communication between tumor cells and their microenvironment, primarily mediated through extracellular vesicles (EVs). These vesicles often contain chemokines such as CCL2, CCL12, and CCL5, which attract immune cells. Once recruited, these immune cells can be reprogrammed by tumor-derived signals to support tumor growth. Pyk2 regulates the trafficking and release of EVs by interacting with Rab GTPases, regulators of intracellular vesicle transport. Through activation of RhoA and reorganization of actin, Pyk2 facilitates vesicle movement toward the plasma membrane and their subsequent secretion. We hypothesized that Pyk2 regulates EV trafficking and secretion in GBM cells by coordinating Rab GTPase activity and actin cytoskeletal remodelling.Primary human GBM cell lines with and without CRISPR/Cas9-mediated Pyk2 knockout (Pyk2KO) were analysed. EVs were isolated and characterized by flow cytometry, MEK/ERK signaling was inhibited using Avutometinib(1µM). Confocal immunofluorescent revealed that Pyk2 promotes Rab27a association with CD63⁺ vesicles, consistent with its role in regulating multivesicular body docking and exosome release. Pyk2 regulates actin dependent trafficking of Rab27a thereby controlling vesicle transport and secretion. Quantitative analysis showed that Pyk2KO shifted the EV population toward larger vesicles. Pyk2KO reduced EV cargo levels of CCL2, CCL5, TNF, and VEGF, mediators of TAM activation and angiogenesis. These data indicate that Pyk2 regulates Rab27a-dependent endosomal vesicle docking and release, whereas MEK/ERK signaling enhances plasma membrane-derived EV shedding. Together, these pathways shape the size and immune regulatory cargo of EVs. Pyk2 controls Rab proteins by vesicle trafficking in glioblastoma cells